Paula M. Martikainen

TMPRSS2:ERG Fusion Identifies a Subgroup of Prostate Cancers with a Favorable Prognosis

Purpose: Our aim was to assess the frequency of ERG overexpression and TMPRSS2:ERG rearrangement in prostate cancer and their association with clinicopathologic variables and outcome. Experimental Design: The presence of the TMPRSS2:ERG rearrangement was studied by reverse transcription-PCR and fluorescence in situ hybridization in 19 prostate cancer xenografts and 7 prostate cancer cell lines. The expression of ERG was studied in the xenografts and cell lines and in 49 freshly frozen clinical prostate samples by quantitative reverse transcription-PCR. The frequency of the TMPRSS2:ERG fusion in clinical prostate cancer (n = 253) on tissue microarrays was assessed by three-color fluorescence in situ hybridization. Results: Seven of 19 (37%) of the xenografts overexpressed ERG and had TMPRSS2:ERG rearrangement. Two xenografts, representing small cell carcinomas, also contained the fusion but did not express ERG. In clinical tumor specimens, the overexpression of ERG was associated with the rearrangement (P = 0.0019). Fifty of 150 (33%) of the prostatectomy specimens and 28 of 76 (37%) of the hormone-refractory prostate cancers on the tissue microarrays carried the TMPRSS2:ERG rearrangement. It was associated with longer progression-free survival in patients treated by prostatectomy (P = 0.019), and according to multivariate analysis, it was an independent predictor of favorable outcome (relative risk, 0.54; 95% confidence interval, 0.30-0.98). The fusion was not associated with Gleason score, pT stage, diagnostic prostate-specific antigen, or cell proliferation activity in prostatectomy specimens nor with the AR gene amplification in hormone-refractory tumors. Conclusions: The TMPRSS2:ERG rearrangement can be found in about one third of prostate cancers. A subgroup of prostate cancer patients with a good prognosis may be identified by the rearrangement.

The Gene for Polycomb Group Protein Enhancer of Zeste Homolog 2 (EZH2) is Amplified in Late-Stage Prostate Cancer

Overexpression of the polycomb group protein enhancer of zeste homologue 2 (EZH2) has been found in several malignancies, including prostate cancer, with an aggressive phenotype. Amplification of the gene has previously been demonstrated in several malignancies, but not in prostate cancer. Our goal was to evaluate the gene copy number and expression alterations of EZH2 in prostate cancer. The copy number of EZH2 in cell lines (LNCaP, DU145, PC‐3, 22Rv1), xenografts (n = 10), and clinical tumors (n = 191) was studied with fluorescence in situ hybridization. All cell lines had a gain of EZH2. Eight of the ten xenografts showed an increased copy number of the gene, including one case of high‐level amplification (≥5 copies of the gene and EZH2/centromere ratio ≥2). 34/125 (27%) of untreated prostate carcinomas showed increased copy number, but only one case of low‐level amplification (≥5 copies of the gene and EZH2/centromere ratio <2), whereas half (25/46) of the hormone‐refractory carcinomas showed increased copy number, including seven cases of low‐level amplification and three cases of high‐level amplification (P < 0.0001). Expression of EZH2 was significantly (P = 0.0009) higher in hormone‐refractory prostate cancer compared with that in benign prostatic hyperplasia or untreated cancer, according to quantitative real‐time RT‐PCR assay. Also, the expression of EZH2 protein was found to be higher in hormone‐refractory tumors than in hormone‐naïve tumors by immunohistochemistry. The EZH2 gene amplification was significantly (P < 0.05) associated with increased EZH2 protein expression. The data show that amplification of the EZH2 gene is rare in early prostate cancer, whereas a fraction of late‐stage tumors contains the gene amplification leading to the overexpression of the gene, thus indicating the importance of EZH2 in the progression of prostate cancer.

EZH2, Ki-67 and MCM7 are prognostic markers in prostatectomy treated patients.

The aim of the study was to evaluate the prognostic value of Ki‐67, EZH2, MCM7 and EIF3S3 in prostatectomy treated patients. A retrospective population‐based material of 249 radical prostatectomy specimens on tissue microarrays was utilized. The median follow‐up of the patients was ∼5.5 years and the main end‐point was biochemical progression. The expression of Ki‐67, EZH2 and MCM7 was determined by immunohistochemistry and the gene copy number of EIF3S3 was analyzed by fluorescence in situ hybridization (FISH). In the whole material, increased immunostainings of EZH2, MCM7 and Ki‐67 were significantly associated with a high Gleason score and a short progression‐free survival. In multivariate analysis, MCM7 and Ki‐67 showed independent prognostic value with relative risks (RR) of 2.65 (95%‐confidence interval of 1.22–5.70), and 1.85 (1.14–3.01), respectively. In subgroup analysis of patients, whose treatment was evaluated to be truly radical (n = 226), EZH2 (3.14, 1.38–7.16), MCM7 (2.70, 1.16–6.30) and PSA (1.5, 1.03–2.20) showed independent prognostic value. In subgroup analysis of cases with a Gleason score <7, low Ki‐67 staining was associated with favorable prognosis with RR of 0.09 (0.01–0.69). In conclusion, Ki‐67, EZH2 and MCM7 are potential prognostic biomarkers in prostatectomy treated patients.

Dual-specificity phosphatase 1 and serum/glucocorticoid-regulated kinase are downregulated in prostate cancer

Inactivation of tumor suppressor genes through deletion, mutation and epigenetic silencing has been shown to occur in cancer. In our study, we combined DNA demethylation and histone deacetylation inhibition treatments with suppression subtraction hybridization (SSH) and cDNA microarrays to identify potentially epigenetically downregulated genes in PC‐3 prostate cancer cell line. We found 11 genes whose expression was upregulated after relieving epigenetic regulation. Expression of 3 genes [dual‐specificity phosphatase 1 (DUSP1), serum/glucocorticoid regulated kinase (SGK) and spermidine/spermine N1‐acetyltransferase (SAT)] was subsequently studied in clinical sample material using real‐time quantitative RT‐PCR and immunohistochemistry. The DUSP1 and SGK mRNA expression was lower in hormone‐refractory prostate carcinomas compared to benign prostate hyperplasia (BPH) or untreated prostate carcinomas. BPH, normal prostate and high‐grade prostate intraepithelial neoplasia (PIN) expressed high levels of DUSP1 and SGK proteins. Ninety‐two percent and 48% of the prostate carcinomas showed almost complete lack of DUSP1 and SGK proteins, respectively, indicating common downregulation of these genes. The genomic bisulphite sequencing did not reveal dense hypermethylation in the promoter regions of either DUSP1 or SGK. In conclusion, the data suggest that downregulation of DUSP1 and SGK is an early event and could be important in the tumorigenesis of prostate cancer.

Second Round Results of the Finnish Population-Based Prostate Cancer Screening Trial

Purpose: Large randomized trials provide the only valid means of quantifying the benefits and drawbacks of prostate-specific antigen (PSA) screening, but the follow-up of ongoing studies is still too short to allow evaluation of mortality. We report here the intermediate indicators of screening efficacy from the second round of the Finnish trial. Experimental Design: The Finnish trial, with ∼80,000 men in the target population, is the largest component in the European Randomized Study of Screening for Prostate Cancer. The first round was completed in 1996–1999. Each year 8,000 men 55–67 years of age were randomly assigned to the screening arm, and the rest formed the control arm. Men randomized to the screening arm in 1996 were reinvited 4 years later, in 2000, and PSA was determined. Results: Of the eligible 6415 men, 4407 (69%) eventually participated in the second round of screening. Of the first-round participants, up to 84% (3833 of 4556) attended rescreening. A total of 461 screenees (10.5%) had PSA levels of ≥4 μg/liter. Altogether, 97 cancers were found, yielding an overall detection rate of 2.2% (97 of 4407). Seventy-nine cases were found among the 3833 second-time screenees (detection rate 2.1%) and 18 in those 574 men (3.1%) who had not participated previously. A PSA of ≥4 μg/liter, but negative biopsy in the first screening round was associated with an up to 9-fold risk of cancer in rescreening relative to those with lower PSA levels at baseline. Ninety-one (94%) of all of the detected cancers were clinically localized. Conclusions: As surrogate measures of an effective screening program, both compliance as well as the overall and advanced prostate cancer detection rates remained acceptable. Men defined as screen-positive but with a negative confirmation of cancer at prevalence screen formed a high-risk group at rescreening.

Membranous location of EGFR immunostaining is associated with good prognosis in renal cell carcinoma

Epidermal growth factor receptor (EGFR) is a key factor in tumorigenesis. The association between EGFR expression and prognosis in renal cell carcinoma (RCC) is not clear. In our study of 134 RCCs, the cellular location of immunostaining was evaluated and patients with EGFR-positive tumours with prominent membranous staining had a good prognosis. Their overall survival was significantly longer (P=0.004) than that of patients with either EGFR-negative tumours or with mainly cytoplasmic staining. However, further studies on the different EGFR expression patterns in RCC are needed to clarify their role in the progression of the disease.

Overexpression and gene amplification of BAG-1L in hormone-refractory prostate cancer.

BAG‐1L (Bcl‐2‐associated anthanogene 1) has been found to interact with androgen receptor (AR), and has been suggested to be involved in the development of prostate cancer. In order to determine the presence of genetic and/or expression alterations of BAG‐1L in prostate cancer, we analysed human prostate cancer cell lines and xenografts as well as patient samples of untreated, hormone‐naïve, and hormone‐refractory prostate carcinomas for sequence variations using denaturing high‐performance liquid chromatography (DHPLC), for gene copy number using fluorescence in situ hybridization (FISH), and for expression using both quantitative RT‐PCR and immunostaining. Only one sequence variation was found in all 37 cell lines and xenografts analysed. BAG‐1 gene amplification was detected in two xenografts. In addition, gene amplification was found in 6 of 81 (7.4%) hormone‐refractory clinical tumours, whereas no amplification was found in any of the 130 untreated tumours analysed. Additionally, gain of the BAG‐1 gene was observed in 27.2% of the hormone‐refractory tumours and in 18.5% of the untreated carcinomas. In a set of 263 patient samples, BAG‐1L protein expression was significantly higher in hormone‐refractory tumours than in primary tumours (p = 0.002). Altogether, these data suggest that amplification and overexpression of BAG‐1L may be involved in the progression of prostate cancer. Copyright

Oxidative/nitrosative stress and peroxiredoxin 2 are associated with grade and prognosis of human renal carcinoma

Peroxiredoxins (Prxs) 1–6 were assessed in 138 renal cell carcinomas (RCC) using immunohistochemistry and selected samples by Western blotting analysis. Oxidative/nitrosative damage was evaluated using nitrotyrosine immunoreactivity. The expressions of Prxs were correlated with tumor grade and survival and nitrotyrosine reactivity. Non‐malignant kidney tubular cells showed positivity with variable intensity for all six Prxs. In RCCs, most cases were positive for Prxs 1 and 2, while only 15–20% of tumors showed expression for Prxs 3 and 4. Prx 2 was associated with tumors of a lower grade (p=0.009) and with a lower frequency of distant metastases (p=0.046). Patients with tumors expressing Prx2 had better prognosis (p=0.027). Instead, nitrotyrosine was significantly associated with high grade tumors (p=0.001). Compared with the non‐malignant kidney tubular cells, low Prx expression in the tumor cells can make them more susceptible to oxidative damage. Prx 2 was more abundantly expressed in low grade tumors, suggesting that this protein could play a role in preventing the development of oxidative damage, which in turn can lead to the activation of pathways leading to aggressive tumors.

Specificity of serum prostate-specific antigen determination in the Finnish prostate cancer screening trial

Specificity constitutes a component of validity for a screening test. The number of false-positive (FP) results has been regarded as one of major shortcomings in prostate cancer screening. We estimated the specificity of serum prostate-specific antigen (PSA) determination in prostate cancer screening using data from a randomised, controlled screening trial conducted in Finland with 32 000 men in the screening arm. We calculated the specificity as the proportion of men with negative findings (screen negatives, SN) relative to those with negative and FP results (SN/(SN+FP)). A SN finding was defined as either PSA⩽4 ng ml−1 or PSA 3.0–3.9 ng ml−1 combined with a negative ancillary test (digital rectal examination, DRE or free/total, F/T PSA ratio). False positives were those with positive screening test followed by a negative diagnostic examination. Of the 30 194 eligible men, 20 794 (69%) attended the first screening round and 1968 (9.5%) had a screen-positive finding. A total of 508 prostate cancers were detected at screening (2.4%). Hence, the number of SN findings was 18 825 and the number of FP results 1358. Specificity was estimated as 0.933 (18 825 out of 20 183) with 95% confidence interval (CI) 0.929–0.936. Specificity decreased with age. Digital rectal examination as ancillary examination had similar or higher specificity than F/T PSA. In the second screening round, specificity was slightly lower (0.912, 95% CI 0.908–0.916). The specificity of PSA screening in the Finnish screening trial is acceptable. Further improvement in specificity could, however, improve acceptability of screening and decrease screening costs.

The Finnish trial of prostate cancer screening: where are we now?

P. FINNE*†, U.-H. STENMAN*, L. MÄÄTTÄNEN‡, T. MÄKINEN§, T.L.J. TAMMELA§, P. MARTIKAINEN¶, M. RUUTU**, M. ALA-OPAS**, J. ARO**, P.J. KARHUNEN††, J. LAHTELA‡‡, P. RISSANEN†, H. JUUSELA§§, M. HAKAMA†‡ and A. AUVINEN† *Department of Clinical Chemistry, University of Helsinki, Helsinki, †School of Public Health and ††Department of Forensic Medicine and Research Unit of Clinical Chemistry, University of Tampere, ‡Finnish Cancer Registry, Helsinki, §Division of Urology, ¶Department of Pathology, Centre for Laboratory Medicine, and ‡‡Department of Internal Medicine, Tampere University Hospital, Tampere, and **Departments of Urology, and §§Surgery, Jorvi Hospital, Helsinki University Central Hospital, Helsinki, Finland